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Systemic lupus erythematosus (SLE) is characterized by the presence of a wide variety of autoantibodies directed against nuclear, cytoplasmic and cell membrane autoantigens. Several lines of evidence, however, suggest that the variety of autoantibodies in SLE may be more limited than was previously thought; different studies clearly suggest that lupus autoantibodies are able to bind multiple antigens. Cross-reactivity, i.e. the ability to bind multiple antigens with high affinity, is not limited to a single autoantibody specificity, but instead seems to be a general property of lupus autoantibodies, distinguishing them from immunization-induced antibodies. Many studies on cross-reactivity have focused on the autoantibodies present exclusively in SLE, and which are therefore considered to be markers of the disease, such as anti-dsDNA, anti-Sm and anti-ribosomal P protein antibodies. However, cross-reactivity has also been shown to be a feature of SLE autoantibodies that are not strictly disease-specific. The structural basis of cross-reactivity is not always clear. It may be due to the presence of common epitopes on different antigens, or to the specific structure of the antigen binding site that can then accommodate different epitopes. Whether one or more mechanisms of cross-reactivity are involved, the ability of an antibody to bind different antigens directly affects its pathogenic potential.

Among anti-nuclear antibodies, anti-Sm and anti-RNP antibodies are of the utmost importance in clinical practice. Anti-Sm antibodies are directed against 7 proteins (B/B', D1, D2, D3, E, F, G) that constitute the common core of U1, U2, U4 and U5 small nuclear ribonucleoprotein (snRNP) particles; B/B', D1 and D3 are more frequently targeted. Anti-RNP antibodies react with proteins (70 Kd, A, C) that are associated with U1 RNA and form U1snRNP. Anti-Sm and anti-RNP antibodies are directed towards both discontinuous and linear epitopes which are either contained in the protein sequence or are post-translationally modified. The assays to detect anti-Sm and anti-RNP antibodies are counterimmunoelectrophoresis (CIE), immunoblot, and ELISA, based on purified or recombinant proteins or synthetic peptides. Anti-Sm antibodies are detectable in a percentage of SLE patients comprised between 5 and 30%; they are more prevalent in blacks and because of their high specificity for SLE have been included in the serological criteria for diagnosing the disease.Anti-RNP are detectable in 25-47% of SLE patients; high titers of anti-RNP antibodies are diagnostic of mixed connective tissue disorder (MCTD). The measurement of anti-Sm and anti-RNP antibodies is more important in the diagnosis of SLE than in the follow-up of patients. However, anti-RNP antibodies are more prevalent in patients with Raynaud's phenomenon and are associated with milder renal involvement. On the contrary, anti-Sm antibodies are associated with the severity and the activity of renal involvement. The specificity of anti-Sm antibodies, together with epidemiological data, suggest that Epstein-Barr virus infection has the potential to induce anti-Sm antibodies by molecular mimicry.Anti-nuclear antibodies, a hallmark of the systemic autoimmune diseases, include several populations of antibodies with different specificities. Among them, anti-Sm and anti-RNP antibodies are of the utmost importance in clinical practice; in research, the study of the mechanisms inducing their production has opened up new perspectives and helped to elucidate the pathogenesis of autoimmune disorders.

Our understanding of antibody-mediated renal damage has not changed drastically in recent years. However, within this framework a lot of new data emerged to change our way of looking at “old” diseases. For example, APSGN, which in the past has been regarded as a typical CIC-mediated disease, may also show ICs formed in situ. In Goodpasture’s syndrome, once considered to be the classical model of autoantibody-mediated disease, it is now realized that nephritogenic T cells play an important role. Complement, formerly thought to be a pathogenic factor in nephritis, is now believed to be crucial for the protection of immune-mediated renal injury. At the same time, components of the fibrinolytic system play a role as autoantibody targets in renal damage, and a disregulation of this pathway may be a mechanism in different renal diseases.

Subclinical gut inflammation has been described in patients with ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Joint involvement has also been reported related to celiac disease. We investigated IgA antibodies to bovine tissue tranglutaminase (tTg) and IgA and IgG antibodies to human tTg and to Saccharomyces cerevisiae (ASCA) in patients with AS and PsA.We evaluated the frequency of IgA antibodies to bovine tTg, and of IgA and IgG antibodies to human tTg and to ASCA in 43 patients with AS and 75 with PsA. As control groups we considered 79 patients with rheumatoid arthritis (RA) and 78 healthy blood donors.We detected antibodies as follows: IgA antibodies to bovine tTg in 1/43 patients with AS, 3/75 with PsA, 1/79 with RA, and in 9/78 healthy controls; IgA antibodies to human tTg in 1/43 patients with AS, 1/75 with PsA, 1/79 with RA, and in 3/78 healthy controls; IgG antibodies to human tTg in 1/43 patients with AS, 4/75 with PsA, 5/79 with RA, and in 7/78 healthy controls. IgA ASCA were confirmed in 10/43 patients with AS, 7/75 with PsA, 14/79 with RA, and in 7/78 healthy controls; IgG ASCA were present in 5/43 patients with AS, 4/75 with PsA, 8/79 with RA, and in 8/78 healthy controls. No statistically significant difference was observed in the prevalence of IgA or IgG antibodies to bovine and human tTg and in the frequency and in mean level of IgA or IgG ASCA between the studied groups or between each group and healthy controls.Our data fail to show an increased prevalence of autoantibodies associated with celiac and Crohn's disease in patients with AS and PsA.

In situ formation of immune complexes is a well recognized mechanism of renal injury in systemic autoimmune disorders. The identification of intrinsic renal antigens that are targets of nephritogenic antibodies is a field of active investigation. Recently, two proteins expressed in the kidney have been characterized as renal antigens. Alpha-actinin, an actin-binding protein localized in glomerular podocytes, is the major target of nephritogenic anti-DNA antibodies. Alpha-enolase, a glycolytic enzyme, is a target of nephritogenic anti-DNA and non-anti-DNA antibodies.

Summary Antibodies specific for ribosomal P proteins (anti-P) are a hallmark of systemic lupus as anti-DNA antibodies are. It has been reported that anti-P antibodies are more frequently detected in anti-dsDNA positive sera. The aim of the present study was to verify the binding ability of anti-P antibodies towards polynucleotides and nucleosomes. We purified anti-P antibodies from 8 SLE patients' sera and we analysed them by ELISA on plates coated with DNA or nucleosomes. We performed also inhibition experiments in order to verify the specificity of the binding. All the purified anti-P antibodies bound DNA, but some anti-DNA binding activity remained among the non-anti-P antibodies in the flow through. Only half of the purified antibodies bound to nucleosomes, and anti-nucleosomal activity was demonstrated also in non anti-P antibody fraction. The inhibition experiments performed on two anti-P antibodies pointed out that only the homologous antigen inhibited the binding to either P peptide or DNA coated onto the solid phase. These results show that sera in which the two specificities coexist contain antibodies endowed with a double binding ability for DNA and the P peptide.

Anti-DNA antibodies and anti-endothelial cell antibodies (AECA) are often detected in systemic lupus erythematosus (SLE). Anti-DNA antibodies can also bind the membrane of human umbilical vein endothelial cells (HUVEC), but little is known about the presence of AECA in the population of immunoglobulins from SLE sera that do not bind DNA. The aim of this study is to analyse the ability of anti-DNA and non-anti-DNA antibodies from SLE sera to bind endothelial cell antigens and to investigate their pathogenic potential. Both anti-DNA and non-anti-DNA antibodies display AECA activity by immunoprecipitation and flow cytometry and in some patients recognize antigens of identical molecular weight. Complement-dependent cytotoxicity on HUVEC was not detected with either anti-DNA or non-anti-DNA antibodies. Similarly, apoptosis was not induced in HUVEC and HL60 incubated with anti-DNA or non-anti-DNA antibodies, as shown by the DNA hypodiploid content. These data indicate that AECA are highly heterogeneous, as they recognize a wide variety of surface molecules on HUVEC and equally present in anti-DNA and non-anti-DNA antibodies from SLE patients.

Background The pathophysiology of rheumatoid arthritis (RA) is currently viewed as a process that starts with a pathologic activation of the immune system that eventually leads to the clinical manifestation of the disease. Systemic autoimmunity has been defined as one of the specific phases preceding the development of RA.(1) Different risk factors may be relevant for each specific phase in RA disease development. Objectives To analyse putative environmental, genetic and demographic risk factors associated with the development of systemic autoimmunity associated with RA in individuals genetically at risk. Methods This is a prospective cohort study of individuals genetically at risk of developing RA, namely first degree relatives (FDRs) of patients with known active RA. Only individuals without clinical evidence of RA were enrolled, and then followed-up yearly to detect the development of active arthritis. We included in this analysis only individuals with available anti-CCP status (anti-CCP 2.0 or anti-CCP 3.1). We pooled individuals from two separate cohorts, one from Switzerland (N=682) and one from France (N=148). We used logistic regression to analyse univariate and multivariate associations between a positive anti-CCP status and putative risk factors. Results A total of 830 FDRs of RA patients are presented, of which 41 (5%) were anti-CCP positive. FDRs had a mean age of 46 years, 79% female, 61% shared epitope positive, and 24% had a history of at least one episode of swollen joint, either at inclusion or during follow-up. In univariate analysis, a positive anti-CCP status was significantly associated with heavy tobacco smoke (>10 pack-years, Odds Ratio (OR): 2.8, p=0.02), loss of teeth (OR: 1.07, p=0.001), the presence of a swollen (OR: 2.5, p=0.04) or a tender joint (OR: 2.1, p=0.03). In multivariate adjusted analysis, only loss of teeth (OR: 1.06, p=0.005), heavy tobacco smoke (OR: 2.6, p=0.039) and the history of a swollen joints (OR: 1.16, p=0.002) were independently associated with anti-CCP positivity. Furthermore, male sex trended to be protective (OR: 0.39, p=0.089) and the effect of obesity on anti-CCP development tended to differ by sex. In males obesity was associated with an increased risk, while in women obesity was associated with a decreased risk (OR: 4.86 vs. OR: 0.18, p=0.075). No association between anti-CCP and the presence of the shared epitope, alcohol consumption or various joint symptoms was found, which may be explained by limited statistical power. Conclusions In individuals genetically at risk for RA, the development of anti-CCP antibodies was associated with tooth loss, tobacco smoke and the occurrence of a swollen joint. Taken together, these results suggest similar risk factors for the production of anti-CCP antibodies and the development of classifiable RA, suggesting that the occurrence of anti-CCP antibodies is a valid intermediate marker of RA development. References Gerlag DM et al. Ann Rheum Dis 2012. Acknowledgements All investigators and participants within the “SCREEN-RA” and the “Campagne des 1009000” studies. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5178

Background Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Up to 50% of RA patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production [1]. The nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Objectives To characterize the autoreactive specificity at single cell level of synovial B cells isolated from TLS+ RA synovium. Methods Single CD19+ B cells were FACS sorted from fresh synovial cell suspension of 3 TLS+ RA joints. RNA was used to amplify Ig VH and VL genes separately and PCR products were cloned and expressed as recombinant monoclonal antibodies each displaying identical specificity of the original B cell [2]. We obtained 139 individual VH sequences (33% IgM, 40% IgG, 27% IgA) and 175 VL sequences. Recombinant monoclonal antibodies were generated from a total of 66 matching H+L chains. Monoclonal antibodies were first screened for their immunoreactivity using a RA autoantigen microarray platform [3]. Immunoreactivity was then confirmed by Luminex, ELISA and WB towards specific citrullinated antigens and by cell-based immunoassays. 30 monoclonal antibodies derived from peripheral naive or memory B cells of patients with primary Sjogren9s syndrome were used as controls. Results VH and VL mutational analysis demonstrated evidence of antigen-driven selection and intra-synovial clonal diversification. Interestingly, IgA clones displayed the highest mutational load followed by IgG and IgM. Among >300 RA autoantigens, the strongest and most frequent immunoreactivity was observed towards citrullinated histones H2A and H2B (∼30% of all RA B cell clones), followed by citrullinated vimentin. Selective reactivity towards the citrullinated but not unmodified forms of H2A, H2B and H4-derived histone peptides was confirmed by ELISA. The intensity of reactivity was directly correlated on the degree of the VH mutations and was abrogated by reverting hypermutated clones to germline sequences by reverse PCR. Citrullinated histones and vimentin are generated during neutrophil extracellular traps (NETs) formation. Indeed, WB on NET protein extracts confirmed selective reactivity towards a 14kDa band corresponding to histones H2/H4. Furthermore, immunofluorescence on neutrophils undergoing PMA-induced NETosis demonstrated specific reactivity of RA clones towards NETs but not resting neutrophils. Conclusions Here we provide evidence for the first time that highly mutated, locally differentiated B cells isolated from RA synovial germinal centre-like structures display strong immunoreactivity towards citrullinated proteins generated during NETosis, primarily citrullinated histones. Thus, synovial NETs emerge as the main antigenic force driving in situ autoreactive B cell activation, selection and differentiation within ectopic germinal centre-like structures sustaining the humoral autoimmune response within the RA joints. References Humby F et al., PLoS Med, 2009. Wardemann H et al., Science, 2003 Robinson, WH et al., Nat Med, 2002. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4422

Background Rheumatoid arthritis (RA) sera contain antibodies specific for deiminated peptide/proteins (ACPA) directed against endogenous (filaggrin, fibrin, vimentin, collagen, enolase) or exogenous antigens. Recently ACPA reacting with citrullinated peptides derived from histone H4 have been detected. It is widely accepted that HLA-DRB1 alleles encoding the shared epitope sequences are associated with the production of ACPAs. However, the genetic background underlying the production of single ACPA specificities has not been thoroughly investigated. Objectives To analyze the reactivity to two different histone H4 derived citrullinated peptides (HCP1 = citrullinated H4 14–34 and HCP2 = citrullinated H4 31–50 ) and to study their association with genetic variants within HLA-DRB1 SE alleles in a RA population. Methods One hundred and seventy-two French RA patients were characterized in terms of HLA-DRB1 genotype, anti VCP-A and anti-VCP-B antibodies. HLA-DRB1 typing was performed using the polymerase chain reaction-sequence specific primer (SSP) method, and SE alleles were classified into four SE+ groups (S1, S2, S3P, S3D) and one SE- group (X) according to the new classification of HLA-DRB1 alleles, reshaping the shared epitope (SE) hypothesis [1]. Anti-HCPs antibodies were assessed by home made ELISA on HCP1 and HCP2 coated plates. Results Setting the threshold value at the 98° percentile of the normal population, anti-HCP1 antibodies are present in 46% and anti-HCP2 antibodies in 62% of RA patients. Anti-HCP1 antibodies are not associated with HLA-SE alleles. On the contrary, anti-HCP2 antibodies are associated with HLA-DRB1 *0102 (p=0.04), *0401 (p=0.03), *0404 (p=0.01). Subgrouping the SE alleles according to the new classification, the presence of anti-HCP2 antibodies is associated with S2 allele (p Conclusions Using two histone H4 derived citrullinated peptides as probes, we showed genetic heterogeneity in the control of the immune response to single components of the ACPA family and to different portion of the same molecule. These data underline the need to type RA patients for different ACPA specificities, that may contribute to the identification of different subsets of patients. References du Montcel ST et al. Arthritis Rheum. 2005, 52(4): 1063-8 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5450

Background Psoriatic arthritis (PsA) is an inflammatory arthropathy associated with psoriasis, characterised by synovitis which often leeds to destruction of cartilage and bone. The mechanism of joint damage is unclear, but it’s likely that cytokines play critical roles. In PsA cytokines have been previously measured by enzyme-linked immunoassays; however, these molecules are not very stable in inflammatory fluids and are detectable only when produced in large amount. Objectives The aim of our study was to analyse the cytokines production in synovial fluids of patients affected by psoriatic arthritis. Methods We analysed cytokines production ex vivo by single cell analysis in flow cytometry in synovial fluids of 9 patients affected by PsA. As control group we used synovial fluids from 8 patients affected by Rheumatoid Arthritis (RA). Synovial fluid leukocytes isolated on Ficoll gradients were stimulated with phorbol-myristate-acetate and ionomycin, treated with brefeldin, permeabilized and stained with anti-CD3, anti-IL4 and anti-IFNg. Results In 9 patients affected by PsA, IFNg-producing cells were the vast majority (43%, 46%, 75%, 55%, 51%, 42%, 38%, 32%, 20% of CD3+ cells). A similar predominance was observed in 8 patients with rheumatoid arthritis (64%, 36%, 35%, 75%, 38%, 29%, 46%, 26%). Conclusion IFNg is the typical cytokine produced by TH1 T cells, thus the results of our study suggest that TH1 T cell selectively home to synovial tissue in PsA, as already shown in RA. A better understanding of cytokine production in psoriatic arthritis synovium in a larger number of patients may help to clarify the pathogenesis of the disease and lead to the development of more specific and effective therapeutic agents. Reference Nuti S, Rosa D, Valiante NM, Saletti G, Caratozzolo M, Dellabona P, Barnaba V, Abrignani S. Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for Va24+ Tcells and rapid elimination of effector cells by apoptosis. Eur J Immunol. 1998;28:3448–56

Rheumatoid arthritis (RA) linkage has been observed on the 18q chromosome in the white population.1 The 18q locus has also shown evidence of linkage with type I insulin-dependent diabetes mellitus, systemic lupus erythematosus and Grave’s disease.2 A candidate gene on chromosome 18q, the TNFRSF11A ( RANK ) gene, which encodes the receptor activator of nuclear factor kappa B, has been shown to be involved in the osteoclast differentiation playing a pivotal role in bone resorption.3 A single RANK haplotype was reported to be associated with RA in a case–control association study.4 Furthermore, activating mutations in the RANK gene cause bone disorders such as early-onset Paget’s disease,5 expansile skeletal hyperphosphatasia6 and familial expansile osteolysis.7 The aim of this …

Introduction ELISA assays Immunoblotting Immunoprecipitation Immunofluorescence Immunohistochemistry Antibody specificity analysed by peptides synthesised on cellulose membranes Appendices.

SUMMARYIn order to analyse the specificity of human anti-ribosomal P protein antibodies, anti-ribosomal P protein antibodies were affinity-purified from the sera of lupus patients. Their binding capacity towards recombinant SmD protein and recombinant SmB/B′ protein was evaluated by immunoblot and ELISA. Epitope mapping of SmD was performed by means of synthetic peptides. Anti-ribosomal P protein antibodies bound recombinant SmD (5/10) and SmB/B′ (4/10) on immunoblot; 6/10 showed binding capacity to SmD on ELISA. Inhibition experiments using ELISA confirmed the specificity of this binding. Our data indicate the cross-reactivity of spontaneously developed anti-ribosomal P protein antibodies with the B/B′ and D constituents of the Sm complex. The coexistence of anti-Sm and anti-ribosomal antibodies in lupus sera may therefore be due, at least in part, to the reactivity of a single autoantibody population.

Renal involvement is one of the most serious manifestations of systemic autoimmune disorders such as systemic lupus erythematosus (SLE) and mixed cryoglobulinemia (MC). The antigen target of the nephritogenic humoral immune response can be classified as renal specific antigens; circulating antigens that can be trapped in the kidney (for example because of charge); soluble antigens that are bound by circulating antibodies. Antibodies that bind renal specific antigens or circulating antigens trapped in the kidney form in situ immunocomplexes. Antibodies that bind soluble antigens present in the vascular compartment form circulating immunocomplexes that can deposit in glomeruli, in basal membrane or mesangium, as well as in vessel walls, tubular basal membrane and interstitium. Deposition of circulating immune complexes has been suggested to play a major role in inducing renal damage in systemic autoimmune disorders. However, a number of studies indicate that autoantibodies present in SLE sera can bind antigens of glomerular cells or constituents of basal membranes. The aim of the present study was to analyze the specificity of antibodies reactive with renal antigens in SLE and MC. MATERIALS AND METHODS

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis. HLA , which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA ( P < 2.5⋅10 −5 ) and nominal for 19 markers in 14 other regions ( P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13 , and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22–23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly ( P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0.001 and suggested an interaction between this putative RA locus and HLA . This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

Background and Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Moreover, RA patients are characterised by increased neutrophils in their synovial fluid, in particular at early stage of the disease. Recent evidence suggests a critical role of neutrophils in sustaining the inflammatory response in the RA joint. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production. However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterised the autoreactive B cell response of lesional B cells isolated from TLS + RA synovium. Materials and Methods Single CD19 + B cells were FACS sorted from synovial cell suspension of 4 TLS + RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform. Results We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H + L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, when the synovial monoclonal antibodies were tested on neutrophil extracellular traps (NETs), which contained citrullinated histones, reactivity towards NETs proteins but not neutrophil nuclear antigens was observed. Conclusions Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones which likely derived from neutrophil NETs that continuously form in the RA synovial fluid. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.

Background Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis, classified within the familyof spondyloarthropathies. The events underlying the pathogenesis of PsA have not yet been fully elucidated. It is known that angiogenesis is an early event in the joint inflammation during PsA, resulting in cartilage degradation and bone destruction. Previous studies have demonstrated an increase of circulating pro-angiogenic mediators, including vascular endothelial growth factor (VEGF) in PsA. The role of endogenous inhibitors of angiogenesis in PsA is unknown. Objectives The aim of our study is to investigate the potential involvement of Endostatin (ES) and Thrombospondin-1 (TSP-1), extracellular matrix (ECM) fragments with anti-angiogenic activity, in patients with PsA. Methods Fifty four adult patients with PsA were studied. The diagnosis of PsA was established according to the CASPAR criteria. Endostatin (ES) and Thrombospondin-1 (TSP-1) were measured, by enzyme immunoassays, in the sera of these patients and of 52 patients with Rheumatoid Arthritis (RA), diagnosed according to the 2010 ACR-EULAR classification criteria, and of 39 healthy subjects. Results The serum level of ES resulted significantly higher in the PsA group than in the control subjects (P Conclusions The observed increase of ECM fragments with anti-angiogenic properties suggests a reduction of pro-angiogenic activity in PsA. It is possible that this reduction is due to underlying treatments. Disclosure of Interest None Declared

Background The spondyloarthritis (SpA), including ankylosing spondylitis (AS) psoriatic arthritis, reactive arthritis, arthritis associated with inflammatory bowel disease and “undifferentiated” SpA (uSpA), are characterised by inflammatory back pain (IBP), sacroiliitis, peripheral arthritis, and enthesitis together with an association with HLA B27. Undifferentiated SpA is a diagnosis of exclusion, and can include patients with IBP who may have early ankylosing spondylitis but do not yet fulfil the classification criteria for AS. Angiogenesis, the formation of new capillaries from pre-existing vessels, isan early event in inflammatory joint diseases. Objectives The aim of our study is to investigate the regulation of pro-angiogenic mediators and their endogenous inhibitors in uSpA and AS. Methods Forty one adult patients with uSpA and 9 patients with definitive diagnosis of AS (according to the 1984 modified New York criteria) were studied. Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF-2) and their inhibitors Endostatin (ES) and Thrombospondin-1 (TSP-1) were measured in the sera of these patients and of 39 healthy subjects by enzyme immunoassays. Results The serum levels of VEGF but not FGF-2 resulted significantly higher in the uSpA group than in the control subjects (P=0.0052). ES and TSP-1 were significantly higher in both uSpA (P Conclusions Our results suggest that a disregulation of circulating angiogenic and angiostatic mediators takes place both in uSpA than in AS. Further investigations on angiogenic and angiostatic mediators in a larger number of patients with different subtypes of SpA would be useful to understand better their role in the pathophysiology of this group of disorders. Disclosure of Interest None Declared

Background Histone deimination is a crucial event in cell biology, regulating gene function and contributing to antimicrobial response, through the formation of neutrophil extracellular traps (NETs.) Deiminated proteins are target of ACPA in rheumatoid arthritis. Objectives Aim of the present study is to to investigate the presence in RA sera of a new subset of ACPA, antibodies reactive with deiminated histones. Methods Neutrophils from peripheral blood were stimulated with calcium ionophore; NETosis was induced by phorbol myristate acetate and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were digested with trypsin and subjected to MALDI-TOF analysis, before and after derivatization to detect citrullinated peptides. Results RA sera reacted with an antigen of 11 KDa expressed in nuclei of activated neutrophils, identified as H4. MALDI TOF analysis indicated that this antigen, present also in NETs, is citrullinated. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titer was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA. Disclosure of Interest None Declared

Background Ectopic lymphoid structures (ELS) are preferential sites for Epstein-Barr virus (EBV) latency and reactivation in the brain of multiple sclerosis (1) and are functional sites of autoreactive B cell activation in the synovial tissue of rheumatoid arthritis (RA) patients (2) leading to the in situ production of anti-citrullinated protein/peptide antibodies (ACPA). However, it is unknown whether ELS are responsible for the generation of antibodies against native and/or post-translationally modified EBV proteins. Objectives In this work we aimed: 1) To investigate the presence of anti-VCA, anti-EBNA1, anti-EA and ACPA antibodies in the serum vs synovial fluid (SF) of RA patients; 2) To provide in vivo evidence of the synovial production of antibodies against unmodified and citrullinated EBV-proteins (3) in the HuRA/SCID chimeric model; 3) To investigate whether EBV-infected synovial plasma cells produce ACPA. Methods Anti-EBNA1, anti-VCA, anti-EA and ACPA IgG were measured in paired serum and SF from RA and OA patients. The local production of unmodified and citrullinated (VCP1 and VCP2) anti-EBV antibodies (3) was investigated by screening the serum of 30 SCID mice transplanted with ELS+ vs ELS- RA synovium. Finally, sequential double immunofluorescence for biotinylated citrullinated fibrinogen (BCf), BFRF1 (EBV lytic antigen) and CD138 was used to detect EBV reactivation in ACPA-producing synovial plasma cells. Results Serum and SF levels of anti-EBNA1, anti-VCA and ACPA IgG were significantly correlated. Sera of SCID mice transplanted with ELS+ (but not ELS-) RA synovia displayed high levels of human IgG ACPA, anti-EBNA1 and anti-VCA but not anti-EA antibodies. Interestingly, a proportion of SCID mice sera displaying high reactivity against anti-CCP and anti-EBNA1 antibodies were also found positive for anti-VCP1 and VCP2. Finally, in ELS+ RA synovia a subset of locally differentiated ACPA+ plasma cells surrounding ectopic germinal centres displayed EBV lytic infection as detected by reactivity for BFRF1. Conclusions Here we showed that antibodies against unmodified and citrullinated EBV antigens are locally produced in the RA synovium in the presence of ELS and are closely associated with in situ ACPA production. Evidence of ACPA+ plasma cells showing lytic EBV infection strongly supports an important role for EBV in the activation and differentiation of autoreactive B cells within the RA synovium. References Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, Andreoni L, Trivedi P, Salvetti M, Faggioni A, Aloisi F. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med. 2007 Nov 26;204(12):2899-912. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C. Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med. 2009 Jan 13;6(1):e1 Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 2006 Mar;54(3):733-41. Disclosure of Interest None Declared

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein-Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies for viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and 11 OA samples by RT-PCR, in situ hybridisation and immunohistochemistry/immunofluorescence. Synovial production of anti-citrullinated proteins (ACPA) and anti-citrullinated EBV peptides (VCP1/VCP2) antibodies was investigated in situ or in vivo in the SCID/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA, but not OA, synovia as revealed by presence of EBV latent [LMP2A, EBV-encoded small RNA (EBER)] transcripts and EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, ~20% of synovial plasma cells producing ACPA were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies cross-recognised by ACPA. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia is favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium that appears to contribute to local growth and differentiation of ACPA-reactive B cells.

The term ‘cryoglobulin’ was introduced to define a group of proteins which have the common property of forming a reversible precipitate or gel in the cold [1]. Three groups of cryoglobulins have been identified. Type I cryoglobulins are monoclonal, the most common one being immunoglobulin M (IgM), a protein associated with immunoprolifera-tive disorders, such as Waldenstrom’s macroglobulinemia, lymphopro-liferative disorders and multiple myeloma.

SUMMARY The frequency and specificity of antibodies to native and denatured collagens were evaluated in systemic sclerosis (SSc) and in primary Raynaud's phenomenon (PRP) by direct and competitive ELISA. Antibodies reactive with denatured collagen type I (CI) were found in 43% of the SSc sera, and anti-CIV and anti-CV in 31%. In PRP, anti-CI, anti-CIV and anti-CV antibodies were detected in 8% of patient sera. Anti-CI, anti-CIV and anti-CV antibodies reacted with determinants expressed on the native as well as on the denatured molecule. Anti-CI and anti-CIV were cross-reactive; a reactivity with CII and a lower one with CV were detected. Anti-CV antibodies also reacted with CI and CII and, in a smaller proportion of cases, with CIV. Anti-collagen antibodies, affinity-purified from blotted collagen IV and V and cyanogen bromide (CBr)-digested CI, displayed the cross-reactivities shown by inhibition studies on sera. Moreover, antibodies eluted from a CBr fragment of CI reacted with the other CBr fragments as well. These data show that one-third of SSc sera contain antibodies that react with epitopes expressed on native as well as on heat-denatured CI, CII, CIV and CV, and therefore have the potential to bind collagens in vivo.

SUMMARYMixed cryoglobulinaemia (MC) is a disorder characterized by the presence of large amounts of cryoprecipitating IgM-IgG complexes. An immune complex glomerulonephritis develops in one third of all patients, but its occurrence docs not seem related to the amount of cryoglobulins in the sera, nor to their complement-fixing ability. In this study we investigated the presence of IgG antibodies reactive with kidney antigens in 33 MC patients (11 with glomerulonephritis, 22 without renal involvement). A total glomerular extract was run on a 10% acrylamide gel, blotted to nitrocellulose and probed with the patients'sera. Sera from half of the patients without renal involvement reacted with several glomerular antigens whose molecular weight ranged between 200 and 29 kD. In the group with renal involvement, sera from 7/11 patients reacted with an antigen of 50 kD, which is also expressed in thymus, but not in the heart or liver. In a follow-up study of four patients with renal involvement, the amount of serum antibody specific for the 50-kD antigen fluctuated, either spontaneously or in response to therapy. These results show that antibodies specific for glomerular antigens are detectable in MC sera. The immune response against a 50-kD antigen expressed in the kidney and thymus seems to be restricted to a subset of MC patients with renal involvement. Circulating autoantibodies specific for glomerular antigens might contribute to the induction of glomerulonephritis in MC forming immune complexes in situ.

Epstein-Barr virus (EBV) infection is associated with production of autoantibodies. The N-terminal 35-58 sequence of EBNA I, one of the nuclear antigens encoded by EBV, is highly homologous to the C-terminal 95-119 region of the ribonucleoprotein SmD. Autoantibodies specific for SmD are present only in systemic lupus (SLE) sera and are therefore considered a serological marker of SLE. We measured antibodies to the EBNA I 35-58 sequence in EBV-related diseases and in autoimmune disorders. Antibodies to the EBNA I 35-58 peptide were present in 30% of normal sera, 12% Burkitt lymphoma, 22% infectious mononucleosis, 25% rheumatoid arthritis, 38% SLE and 33% Sjogren's syndrome. Antibodies to the SmD 95-119 peptide were detectable in 32% of SLE sera, 17% infectious mononucleosis and 12% Burkitt lymphoma. The specificity of anti-EBNA I 35-58 antibodies affinity-purified from nine sera was analysed by means of an inhibition assay. Only anti-EBNA I 35-58 antibodies affinity-purified from SLE sera have a similar affinity for the viral peptide and the SmD C-terminal one; they also bind the recombinant SmD in western blot. The results indicate that antibodies to EBNA I 35-58 are produced in normals, in EBV-related diseases and in autoimmune disorder, but only SLE sera contain anti-viral antibodies cross-reactive with an autoantigen.

Antibodies against small nuclear ribonucleoproteins (snRNP) are frequently produced in systemic autoimmune disorders and different mechanisms have been proposed to account for their production. Molecular mimicry is a well-established phenomenon which can involve exogenous antigens as well as autoantigens. An example of an exogenous antigen, i.e. a viral protein, was reported by Guldner et al. (1990). A 5-amino acid sequence of the p68 protein, a component of U1 snRNP, is shared with the matrix protein of the influenza B virus. A subset of patients with autoimmune disorders, whose sera contain anti-p68 antibodies, react with this shared sequence and with the entire matrix protein. The same p68 protein bears in another region a strong sequence-homology with the third complementarity-determining region (CDR3) of an immunoglobulin light chain. Immunization of normal animals with the light chain induces the production of anti-p68 antibodies (Puccetti et al, 1990). In this case, the sequence mimicking the ribonucleoprotein is contained in the variable region of the immunoglobulin, where the idiotope is located.

SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C-terminal region of SmD (sequence 95-119). This region is highly homologous to sequence 35-58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein-Barr virus. Antibodies affinity purified over a peptide 95-119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti-SmD 95-119 antibodies bound also the EBNA I 35-58 peptide and detected the EBNA I molecule in a total cell extract from Epstein-Barr virus-infected lines. A population of anti-SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti-SmD antibodies, we immunized mice with the EBNA I 35-58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti-SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95-119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti-SmD autoantibodies.

The immunogenicity of the non-repetitive sequences of the Plasmodium berghei circumsporozoite (CS) protein was studied using synthetic peptides. Two CS sequences (residues 20-39 and 57-70) exhibiting T cell helper activity were identified. Immunization of BALB/c mice with a branched peptide containing either the 20-39 or the 57-70 sequence and two repeats (B epitope) in a linear sequence induced high titers of anti-repeat and anti-sporozoite antibodies. Mice immunized with the T-B construct (high antibody titers) or with the 57-70 epitope alone (no serum anti-repeat or anti-peptide antibodies) were protected to a similar degree after challenge with infective sporozoites. No protection was obtained in mice immunized with the 20-39 epitope. These results indicate that BALB/c mice can be protected either by effector T cells or by high levels of anti-repeat antibodies. Thus, in the same strain, a double mechanism of protection can be obtained by a synthetic peptide vaccine.

Since dietary macromolecular antigens can be involved in the pathogenesis of IgA nephropathy (IgAN), the effect of a low-antigen-content diet was evaluated in 21 patients (10 women, 11 men, mean age 27.7 +/- 10 years) with immunohistochemical findings of active IgAN. The diet was followed for a 14-24-week period (mean 18.8 +/- 6); in all cases the effects of the treatment were evaluated by clinical and serological parameters, and in 11 patients also by repeat renal biopsy. After dietetic therapy a significant reduction of urinary proteins was recorded (P0.001); in particular, heavy proteinuria (1 g/day), present in 12 cases during the 6 months preceding the treatment, was markedly reduced or disappeared in 11. At post-treatment control biopsy mesangial and parietal deposits of immunoglobulins, complement C5 fraction and fibrinogen were significantly reduced. The improvement of the objective parameters such as heavy proteinuria, a strong predictor of a poor prognosis, and of immunohistochemical alterations indicate that a low-antigen diet can positively affect patients with IgAN. These results could be ascribed to a reduction of nephritogenic food antigen input and to a putative functional restoration of the mononuclear phagocytic system.

Macrophage production of arginine-derived NO2- provides a simple method for detection and quantitation of interferon-gamma (IFN-gamma) in murine cell culture fluids. When the macrophage cell line RAW 264 is cultured overnight with IFN-gamma in the presence of 10 ng/ml LPS, NO2- release, as determined by a simple colorimetric assay, is proportional to the concentration of IFN-gamma and is inhibited by monoclonal antibody to IFN-gamma. A high degree of correlation was obtained when antigen stimulated T lymphocyte supernatants were tested for IFN-gamma by ELISA and the NO2- production assay.

The VH and V kappa gene families expressed by 20 monoclonal auto-anti-idiotypes (Ab2) derived from unmanipulated MLR-lpr/lpr mice were determined by Northern blotting. Complete variable region sequences of six Ab2, along with three additional V kappa-JH Ab2 sequences, were obtained. These auto-anti-idiotypes arose spontaneously in the animals, and they bound specifically to an idiotypic determinant (Id/r) on mAb 28/12, a monoclonal IgG2b MLR-lpr/lpr anti-small nuclear ribonucleoprotein antibody. The 16 Ab2 heavy chains belonged to 7 different VH gene families, and the 10 Ab2 light chains were derived from 8 V kappa families. The light chains of two Ab2 were approximately 99% identical; the remaining variable region sequences were highly heterogeneous. There was no correlation between primary amino acid sequence of either heavy or light chain and idiotypic properties of the auto-anti-idiotypes. Six Ab2 used VH or V kappa genes that are identical to known germ-line genes. A high proportion of the spontaneous auto-anti-idiotypes was shown to have autoantibody activity (anti-DNA, anti-ribonucleoprotein), or specific binding reactions with lipopolysaccharide of Salmonella RE, or both properties. The structural diversity of spontaneous MLR-lpr/lpr auto-anti-idiotypes differs sharply from the structural homogeneity reported for Ab2 induced in normal animals against syngeneic Ab1. Our results suggest that auto-anti-idiotypes might arise independently of an immunogenic stimulus from an Ab1.

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.

A number of rheumatoid arthritis (RA) sera contain antibodies specific for peptides in which arginine is substituted by the deiminated form citrulline (AKA). These antibodies are a marker of RA, as they are absent in other disorders. The enzyme responsible for the generation of citrulline residues, peptidylarginine deiminase (PAD), has different isoforms, with a specific tissue distribution. PAD V, expressed in monocytes, might be responsible for the deimination of arginine residues of synovial proteins and thus be involved in the generation of epitopes for RA-specific antibodies. The ECRAF genome scan showed suggestive linkage evidence at PAD V locus on chromosome 1 (P < 0.005). We decided to analyze PAD V as a candidate gene for RA, studying a cohort of 100 RA patients (tested for AKA) and their unaffected parents. Investigation (by single-strand conformation polymorphism [SSCP] analysis and sequencing) of the 16 exons, 5' and 3' regions of the PAD V gene provided polymorphisms in the 5', exons 3, 4, and 7 and 3' regions. Analysis used the transmission disequilibrium test and the haplotype relative risk for alleles and haplotypes with Analyze and Genhunter2 programs. We found an association between RA and one PAD V haplotype (38% in RA versus 17% in controls) (P < 0.007). The association was also observed in the AKA+ RA subgroup (41%) (P < 0.03). In conclusion, the PAD V gene may be considered one of the genetic factors that confer susceptibility to RA. Studies are in progress to clarify the relationship between the PAD V haplotypes, the enzyme activity and the production of anticitrulline antibodies.

The glycolytic enzyme 2-phosphoglyceratelyase (alpha-enolase) is an autoantigen in connective tissue disorders, and more frequently in patients with active renal disease. The enzyme has pleiotropic functions: it is also a structural protein, a stress protein induced by hypoxia and it acts as transcription factor in the nucleus. Alpha enolase is encoded by a single copy gene and only one mRNA species is detected. In order to define a structural basis for these different functions, we analyzed the isoelectric point of the enzyme. On a kidney extract fractionated by 2D electrophoresis, a mouse anti-enolase antiserum detects 5 spots of identical molecular weight but differing in pI. Some autoimmune sera react with all the spots, while other recognize only the acidic forms of alpha-enolase. We then analyzed the properties of the membrane form of enolase. Enolase is not a membrane structural protein, but it is strongly associated with the membrane, where it acts as plasminogen receptor. Anti-enolase antibodies purified from autoimmune sera react also with the membrane form of alpha-enolase: by flow cytometry, 7/9 antibody preparations bind in fact U937 cells, a human lymphomonocytoid cell line that expresses high density of plasminogen receptors. To investigate the possible functional role of membrane enolase, we evaluated the ability of monoclonal anti-enolase antibodies to induce cell damage or apoptosis. No monoclonal had a cytotoxic effect on U937 cells or was able to induce apoptosis in the same cell line. We then tested the ability of monoclonal anti-enolase antibodies to induce Ca2+ influx in U937 cells. One out of 4 monoclonal antibodies induced release of Ca2+ from intracellular stores. In conclusion, alpha-enolase exists as multiple isoforms, probably due to postranslational modifications, which seem to affect recognition by autoantibodies. It is presently unknown whether these modifications are tissue-specific and/or affect membrane expression of the enzyme. A possible link between Ca2+ influx and receptor functions of enolase is currently under investigation.

Objective It has been proposed that noninherited maternal antigens (NIMA) (HLA–DR antigens) might play a role in susceptibility to rheumatoid arthritis (RA), especially in patients who are not genetically predisposed, such as those who are HLA–DR4 and/or shared epitope (SE) negative. The present study was undertaken to test the NIMA hypothesis in a large cohort of European RA patients assembled by the European Consortium on RA Families (ECRAF). Methods HLA–DRB1 oligotyping was performed in families of European RA patients for whom both parents were alive. These families were consecutively recruited by the ECRAF between 1996 and 1998, for association studies. The frequencies of HLA–DR NIMA were compared with those of the noninherited paternal antigens (NIPA) after stratification for HLA–DR*04, *0401 and/or *0404, and SE status. NIMA or NIPA that coincided with inherited HLA–DR antigens were considered redundant and excluded from analysis. Calculations concerning the whole group and restricted to patients lacking parental RA were performed. Results One hundred seventy families from France (n = 81), Belgium (n = 23), Spain (n = 24), Italy (n = 19), Portugal (n = 14), and The Netherlands (n = 9) were oligotyped. The group of probands was predominantly female (88%), positive for rheumatoid factor, DR*04, and SE (71%, 58%, and 75%, respectively), and had erosive disease (75%). Parental RA was reported in 21 families. Using the NIPA as control, the frequency of HLA–DRB1*04, *0401 and/or *0404–, or SE-positive NIMA was not found to be increased in patients lacking these susceptibility alleles. The same was true when the 21 probands with parental RA were excluded from analysis. In DRB1*04-positive patients, we found no evidence of a relevant effect of HLA–DR3 or DR6 in the NIMA. Conclusion Our results do not support the notion that noninherited maternal antigens have a role in susceptibility to RA in the offspring.